DLC coatings for UHMWPE: Relationship between bacterial adherence and surface properties

Development of intrinsically antibacterial surfaces is of key importance in the context of prostheses used in orthopedic surgery. This work presents a thorough study of several plasma-based coatings that may be used with this functionality: diamond-like carbon (DLC), fluorine-doped DLC (F-DLC), and a high-fluorine-content-carbon-fluor polymer (CF(X) ). The coatings were obtained by a radio-frequency plasma-assisted deposition on ultra high molecular weight polyethylene (UHMWPE) samples and physicochemical properties of the coated surfaces were correlated with their antibacterial performance against collection and clinical Staphylococcus aureus and Staphylococcus epidermidis strains. The fluorine content and the relative amount of C?C and C?F bonds were controlled by X-ray photoelectron spectroscopy, and hydrophobicity and surface tension by contact angle measurements. Surface roughness was studied by Atomic Force Microscopy. Additional nanoidentation studies were performed for DLC and F-DLC coatings. Unpaired t test and regression linear models evaluated the adherence of S. aureus and S. epidermidis on raw and coated UHMWPE samples. Comparing with UHMWPE, DLC/UHMWPE was the least adherent surface with independence of the bacterial species, finding significant reductions (p ≤ 0.001) for nine staphylococci strains. Bacterial adherence was also significantly reduced in F-DLC/ UHMWPE and CFx/UHMWPE for six strains. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:2813-2820, 2012.     

Membrane insertion of lymphocyte surface molecules

We have used different radiolabelling procedures, followed by immunoprecipitation and SDS-PAGE†, to assess the membrane disposition of various lymphocyte surface molecules. [¹²⁵I]-lodonaphthylazide labels lipid-associated portions of membrane molecules, whereas lactoperoxidase-catalysed iodination labels protein segments which are exposed on the outside of the plasma membrane. Our results here show that in murine splenocytes, surface Ig μ and δ heavy chains, H-2 heavy chains, and I-A alpha and beta chains are all inserted into the lipid bilayer and exposed on the cell surface. Surface Ig L chains and H-2-associated beta-2-microglobulins are not membrane-inserted, but are bound to the cell surface via association with their respective heavy chains. I-A invariant chains are labelled neither on the cell surface nor within the lipid bilayer, and so may reside in the cytoplasm. With surface Ig δ chains, the F_c rather than the F_d portion is shown to be membrane-inserted. Finally, an unidentified 28K dalton protein, also residing within the lipid bilayer, is shown to be coprecipitated with surface Ig.     

Inhibition of cell surface export of group A streptococcal anchorless surface dehydrogenase affects bacterial adherence and antiphagocytic properties

Surface dehydrogenase (SDH) is an anchorless, multifunctional protein displayed on the surfaces of group A Streptococcus (GAS) organisms. SDH is encoded by a single gene, sdh (gap or plr) that is essential for bacterial survival. Hence, the resulting nonfeasibility of creating a knockout mutant is a major limiting factor in studying its role in GAS pathogenesis. An insertion mutagenesis strategy was devised in which a nucleotide sequence encoding a hydrophobic tail of 12 amino acids ((337)IVLVGLVMLLLS(348)) was added at the 3' end of the sdh gene, successfully creating a viable mutant strain (M1-SDH(HBtail)). In this mutant strain, the SDH(HBtail) protein was not secreted in the medium but was retained in the cytoplasm and to some extent trapped within the cell wall. Hence, SDH(HBtail) was not displayed on the GAS surface. The mutant strain, M1-SDH(HBtail), grew at the same rate as the wild-type strain. The SDH(HBtail) protein displayed the same GAPDH activity as the wild-type SDH protein. Although the whole-cell extracts of the wild-type and mutant strains showed similar GAPDH activities, cell wall extracts of the mutant strain showed 5.5-fold less GAPDH activity than the wild-type strain. The mutant strain, M1-SDH(HBtail), bound significantly less human plasminogen, adhered poorly to human pharyngeal cells, and lost its innate antiphagocytic activity. These results indicate that the prevention of the cell surface export of SDH affects the virulence properties of GAS. The anchorless SDH protein, thus, is an important virulence factor.     

Impact of convective transport on dialyzer clearance

 The results of predictions of three mathematical models used to describe the impact of convective flow on dialyzer clearance are presented. These models are based on the ordinary differential equations, which describe changes of solute concentration and solute and fluid flows along the module length. One of the models takes into consideration the existence of the boundary layers on both sides of the membrane wall, by including in the equations two parameters k _B and k _D , which describe mass transport coefficients in blood and dialysate, respectively. In the second model, the boundary layers are included in one lumped membrane permeability parameter. The diffusive membrane permeability was calculated from pure diffusive clearance, which was taken from experimental results. In the third model, a linear dependence of transmittance coefficient and ultrafiltration flow was proposed. The theoretical results were compared with data obtained in experiments carried out in vitro with four types of high-flux hollow-fiber dialyzers. The comparisons demonstrate that the first two models are of similar accuracy and the third model is not suitable for small solutes. Received: June 17, 2002 / Accepted: October 16, 2002 Acknowledgments This study was supported by Fresenius Medical Care Deutschland GmbH. The authors thank Dr. Cecile Legallais, University of Compiegne, France, for help with the references. Correspondence to:M. Galach     

Incorporation of Si and SiOx into diamond-like carbon films: Impact on surface properties and osteoblast adhesion

The interaction of human osteoblast cells with diamond-like carbon films incorporating silicon and silicon oxide (SiO_x, 1 ? x ? 1.5) and synthesized using the direct-current plasma-activated chemical vapour deposition method was investigated. Cell culture studies were performed for films with Si contents ranging from ～4 at.% to 15 at.%. Substantial differences between Si-incorporated and SiO_x-incorporated films were found for the bonding environments of Si atoms and the hybridization of underlying carbon structures. However, osteoblast-attachment studies did not show statistically significant trends in properties of cell growth (count, area and morphology) that can be attributed either to the Si content of the films or to the chemical structure of the films. The surface energy decreased by 40% as the Si content of the SiO_x incorporated DLC films increased to 13 at.%. The cell adhesion properties however did not change in response to lowering of the surface energy. The incorporation of both Si and SiO_x leads to a beneficial reduction in the residual stress of the films. The average roughness of the films increases and the hardness decreases when Si and SiO_x are added to DLC films. The impact of these changes for load-bearing biomedical applications can be determined only by carefully controlled experiments using anatomic simulators.     

Monoclonal IgA class-switch variants against bacterial surface antigens: molecular forms and transport into murine respiratory secretions

The present study describes a new model for passive immunization of the respiratory tract with IgA in comparison to other isotypes. Monoclonal IgA-isotype-switch variants were isolated from different IgG-producing hybridoma clones specific for surface epitopes of bacterial respiratory tract pathogens. Analysis of the molecular form of the IgA variants revealed the simultaneous production of monomeric, dimeric and higher polymeric IgA by a single-cell line with predominance of the polymeric forms. The specificities of the IgA variants were identical to the parent IgG antibodies as demonstrated by inhibition experiments. The IgA variant antibodies were separated into monomers and polymers by gel filtration. Intravenous injection of the different molecular forms of IgA and of IgG into mice were used to investigate the transport characteristics of IgA into murine upper and lower respiratory tract secretions by the physiological route in comparison to IgG. Polymeric IgA variant, monomeric IgA variant and IgG were detected in immunologically active form in both nasal secretion and bronchoalveolar fluid as evidenced by binding to their antigens in an enzyme-linked immunosorbent assay. The relative contribution of the specific exogenous monoclonal IgA and monoclonal IgG to total IgA and IgG, respectively, was determined in secretions. Comparison of the secretion to serum transport ratios clearly indicates selective transport of polymeric IgA variant into nasal secretions relative to IgG parent antibody. Molecular and functional characteristics of the IgA variants make them ideal for passive mucosal immunization experiments and identification of protective epitopes in mucosal immunity.     

Impact on the thrombogenicity of surface oxide properties of 316l stainless steel for biomedical applications

Surface oxide film on cardiovascular devices could be one of the most critical factors that determine the degree of thrombosis. Previous studies have shown that metallic wire passivated with amorphous oxide film provides excellent corrosion and scratch resistance. Investigation was undertaken to see whether this oxide film, with its unique electrochemical properties, could reduce the development of thrombosis. Results show that amorphous oxide has higher value of time constant, lower open-circuit potential, and lower degree of thrombosis. These distinguished characteristics prove amorphous oxide to be the best candidate for the cardiovascular devices. Amorphous oxide film could be a potential solution to the thrombogenic problem of cardiovascular devices.     

聚氯乙烯材料表面细菌生物膜结构观察

目的 建立聚氯乙烯（PVC）材料表面细菌生物膜（BF）体外模型,并探讨激光共聚焦显微镜（CLSM）和环境扫描电子显微镜（SEM）对研究BF的应用价值. 方法 用生物膜形成阳性的表皮葡萄球菌（表葡菌）RP62A,在TBS培养基中,进行体外PVC材料表面BF形成实验动态观察,分别于培育6、12、18、24、30、48 h后,用CLSM观察BF的厚度、单位面积BF群落数量、断层扫描图像、BF中活和死菌荧光比例及其三维重建图像,用环境SEM观察BF表面精细结构. 结果 表葡菌BF是具有高度组织化的多细胞群体结构,其中细菌密集,由活菌和死菌组成;PVC材料表面BF的形成是动态的过程,12～18 h细菌黏附达到高峰,24 h形成成熟BF;成熟BF中,内层、中间层及外层的活、死菌百分率比较差异有统计学意义（P＜0.05）. 结论 表葡菌生物膜结构复杂,CLSM与环境SEM的有机结合是观察PVC材料表面细菌BF的理想方法.     

The role of the interplay between polymer architecture and bacterial surface properties on the microbial adhesion to polyoxazoline-based ultrathin films

Surface platforms were engineered from poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA) copolymers to study the mechanisms involved in the non-specific adhesion of Escherichia coli (E. coli) bacteria. Copolymers with three different grafting densities α (PMOXA chains/Lysine residue of 0.09, 0.33 and 0.56) were synthesized and assembled on niobia (Nb?O?) surfaces. PLL-modified and bare niobia surfaces served as controls. To evaluate the impact of fimbriae expression on the bacterial adhesion, the surfaces were exposed to genetically engineered E. coli strains either lacking, or constitutively expressing type 1 fimbriae. The bacterial adhesion was strongly influenced by the presence of bacterial fimbriae. Non-fimbriated bacteria behaved like hard, charged particles whose adhesion was dependent on surface charge and ionic strength of the media. In contrast, bacteria expressing type 1 fimbriae adhered to the substrates independent of surface charge and ionic strength, and adhesion was mediated by non-specific van der Waals and hydrophobic interactions of the proteins at the fimbrial tip. Adsorbed polymer mass, average surface density of the PMOXA chains, and thickness of the copolymer films were quantified by optical waveguide lightmode spectroscopy (OWLS) and variable-angle spectroscopic ellipsometry (VASE), whereas the lateral homogeneity was probed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). Streaming current measurements provided information on the charge formation of the polymer-coated and the bare niobia surfaces. The adhesion of both bacterial strains could be efficiently inhibited by the copolymer film only with a grafting density of 0.33 characterized by the highest PMOXA chain surface density and a surface potential close to zero.     

In vitro study of blood-contacting properties and effect on bacterial adhesion of a polymeric surface with immobilized heparin and sulphated hyaluronic acid

The blood-contacting properties and the effect on bacterial adhesion of a material based on polyurethane and poly(amido-amine) (PUPA), both in its native form and with the anticoagulant molecules heparin or sulphated hyaluronic acid (HyalS3.5) electrostatically bonded to its surface, were evaluated and compared in vitro. The presence of the biological molecules on the surface was revealed by a dye test and ATR/FTIR analysis. Bound heparin was found to maintain its physiological action, in terms of thrombin inactivation, as well as did free heparin. Moreover, it reduced the degree of platelet adhesion. On the contrary, bound HyalS3.5 lost its anticoagulant activity, though it reduced platelet adhesion. The number of platelets on both modified surfaces was low. Their shape distribution, as determined by SEM, did not differ significantly on the two modified surfaces or with respect to the bare PUPA surface. HyalS3.5 and heparin also inhibited adhesion of Staphylococcus epidermidis to the material. A possible relationship between the platelet and bacterial adhesion is ascribed to the mediating role of plasma proteins.     

In vitro study of blood-contacting properties and effect on bacterial adhesion of a polymeric surface with immobilized heparin and sulphated hyaluronic acid

The blood-contacting properties and the effect on bacterial adhesion of a material based on polyurethane and poly(amido-amine) (PUPA), both in its native form and with the anticoagulant molecules heparin or sulphated hyaluronic acid (HyalS3.5) electrostatically bonded to its surface, were evaluated and compared in vitro. The presence of the biological molecules on the surface was revealed by a dye test and ATR/FTIR analysis. Bound heparin was found to maintain its physiological action, in terms of thrombin inactivation, as well as did free heparin. Moreover, it reduced the degree of platelet adhesion. On the contrary, bound HyalS3.5 lost its anticoagulant activity, though it reduced platelet adhesion. The number of platelets on both modified surfaces was low. Their shape distribution, as determined by SEM, did not differ significantly on the two modified surfaces or with respect to the bare PUPA surface. HyalS3.5 and heparin also inhibited adhesion of Staphylococcus epidermidis to the material. A possible relationship between the platelet and bacterial adhesion is ascribed to the mediating role of plasma proteins.     

骨科植入物表面细菌生物膜的研究与机制

背景：细菌形成生物膜后耐药性极强,而外科感染特别是骨科植入物感染与植入材料表面形成细菌生物膜密切有关。 目的：综述了细菌生物膜形成、结构、影响因素、耐药机制及防治措施的研究进展。 方法：以 “Bacterial biofilm,Drug resistance,Implant,Infection”为英文检索;以“细菌生物膜,耐药性,植入物,感染”中文检索词,检索 CNKI和PubMed数据库1982年1月至2011年9月有关细菌生物膜及其防治,植入物术后感染形成生物膜相关研究。纳入34篇文献进行分析。 结果与结论：抗生素应用不能有效清除细菌生物膜,还可诱导耐药性产生。改变生物膜的微环境、选择生物相容性好的内植入材料和降低细菌的黏附性可以更加有效控制感染。     

Impact of pathogenicity islands in bacterial diagnostics

Pathogenicity islands (PAIs) are a distinct class of genomic islands (GEIs), which are acquired by horizontal gene transfer. PAIs harbour virulence genes and some, in addition, antibiotic resistance genes. More often genes conferring antibiotic resistance are encoded by GEIs not containing virulence genes. Both types of genetic elements are found in genomes of various human, animal and plant pathogens. There are PAIs and GEIs which are specific for a certain serotype(s), strain, or pathotype of a species. Furthermore, there are also PAIs which are more widespread and found in bacterial pathogens causing a certain pathogenic effect in the host. Even the lack of a certain PAI might be characteristic for a defined subspecies. Obviously, PAIs can be used as markers for diagnostic purposes to help identify a certain bacterial pathogen, subtype it, estimate the pathogenic potential, and in some cases predict its antibiotic resistance. This all might be achieved for known PAIs/GEIs without cultivating the microorganism of interest by employing PCR and/or DNA-chip technology. Even yet unknown PAIs can be identified in silico if the genome sequence of the bacterial pathogen under investigation is known. The more PAIs and antibiotic harbouring GEIs are identified and characterized the greater will be the benefits also for diagnostics.     

Effect of surface chemistry on bacterial adhesion, viability, and morphology

Helicobacter pylori (H. pylori) is one of the most common infectious agents in the world and it is thought to colonize the gastric mucosa of about half of the world's population causing several gastric diseases. In this work, the effect of surface chemistry on H. pylori nonspecific adhesion, viability, and morphology was evaluated using three H. pylori strains with different adhesins expression profile. Self-assembled monolayers (SAMs) of alkanethiols on gold were used to obtain surfaces exposing different functional groups: OH, CH3, and ethylene glycol (EG4). Bacterial adhesion onto the surfaces reached a plateau at 2 h. There was a correlation between adhesion and the exposed surface group, with bacterial cells adhering preferentially to CH3-SAMs while EG4-SAMs prevented H. pylori adhesion during the entire adhesion test (24 h). Surfaces that presented the EG4 group were also the only ones that significantly reduced the viability of adhered bacteria. Surface chemistry also influenced the morphology of adhered bacteria. The H. pylori rod shape observed in the control (Tissue Culture Polyethylene-TCPE) was only retrieved on CH3-SAMs. This work demonstrates that surface chemistry, namely specific functional groups on the material, influence the nonspecific adsorption of H. pylori. Moreover, the features of the bacterial strain and the surface chemistry can alter the adhesion kinetics, as well as the morphology and viability of attached bacteria.     

Structure and properties of animal and bacterial collagenases

Everywhere is found collagen, collagenases are also found. The author reviews the properties of these collagenases: structure, biosynthesis, activity, inhibition. The therapeutical applications are also discussed. They seem promising since a collagenase I, Achromase can be industrially produced and has been shown interesting both in laboratory studies and in clinical trials.